Lipid vesicles containing adeno-associated virus rep protein for transgene integration and gene therapy

ABSTRACT

A composition for delivering at least one DNA sequence encoding a desired protein or polypeptide (such as a therapeutic agent) to a cell. The composition comprises an adeno-associated virus rep protein (or a nucleic acid sequence encoding an adeno-associated virus rep protein) and a genetic construct including at least one DNA sequence encoding a protein or polypeptide or genetic transcript of interest and a promoter controlling the at least one DNA sequence. The genetic construct also includes a first adeno-associated virus ITR or protein or derivative thereof and a second adeno-associated virus ITR or a portion or derivative thereof. The first and second adeno-associated virus ITRs or portions or derivatives thereof flank the at least one DNA sequence encoding the protein or polypeptide or genetic transcript of interest and the promoter controlling the at least one DNA sequence encoding the protein or polypeptide or genetic transcript of interest. Such a composition provides for integration of genetic material at a specific locus in the human chromosome, while minimizing the possibility of inadvertent inactivation of host genes and minimizing the possibility of viral contamination.

This invention relates to gene transfer wherein a desired gene isdelivered to a eukaryotic cell with applications for gene therapy. Suchgene delivery may be accomplished in vivo, or may be accomplished invitro, followed by the in vivo administration of such eukaryotic cellsto a host. More particularly, this invention relates to liposomes andsimilar transfection vehicles which include an adeno-associated virusrep protein, adeno-associated virus ITRs, and DNA encoding a desiredprotein, polypeptide or genetic transcript, such as messenger RNA,antisense RNA, or a ribozyme.

BACKGROUND OF THE INVENTION

Adeno-associated virus (or AAV) has the unique ability to target theintegration of its DNA into a host cell genome in a non-random,locus-specific manner. This is in contrast to other viruses such asretroviruses which integrate at random positions in the host genome.

The left open reading frame of adeno-associated virus encodes the repproteins. Two promoters located at map positions 5 and 19 (promoters p5and p19, respectively) control expression of the four proteins derivedfrom this ORF. Rep proteins Rep 78 and Rep 68 are produced from p5promoted transcripts, and rep proteins Rep 52 and Rep 40 are producedfrom p19 promoted transcripts. It has been demonstrated in vitro thatthe p5 promoted rep proteins (rep 78 and Rep 68) bind to a definedregion of human chromosome 19 at the integration locus for AAV provirus.

It is therefore an object of the present invention to employ AAV repprotein and the AAV ITRs as part of a gene delivery system for achievingtargeted integration of foreign genes. Such targeted integration wouldprovide a more effective and safer method of gene delivery. Other genedelivery techniques achieve low levels of integration, often requireactively cycling cells as targets, and if integration occurs, it happensat random sites in the genome. Random integration poses the potentialdanger of inadvertent activation of a deleterious gene (such as aprotooncogene) or inadvertent inactivation of an essential gene.

Many clinical gene therapy experiments or protocols also employviral-based gene delivery systems. Such procedures pose the risk ofcontamination with potentially pathogenic wild-type virus, which is asignificant safety concern. Also, these systems may result insignificant host immune responses to transfected cells that expressviral proteins on their surfaces.

BRIEF DESCRIPTION OF THE FIGURES

The invention now will be described with respect to the figures,wherein:

FIG. 1 is a map of plasmid AAVp5neo;

FIG. 2 is a map of plasmid pSv-β-galactosidase;

FIG. 3 is a map of plasmid TRF169;

FIG. 4 is a map of plasmid pLZ11;

FIG. 5 is a map of plasmid pSP72;

FIG. 6 is a map of plasmid pSP72nlacZ;

FIG. 7 is a map of plasmid pAdRSV4;

FIG. 8 is a map of plasmid pAdRSVnlacZ;

FIG. 9 is a map of plasmid pAAVrnlacZ;

FIG. 10 is a map of plasmid pPR997;

FIG. 11 is a map of plasmid pMBP-Rep 68Δ;

FIG. 12 is a map of plasmid pMBP-Rep 68ΔNTP

FIG. 13 is a map of plasmid pMBP-Rep 78;

FIG. 14 is a map of plasmid pAv1H9FR;

FIG. 15 is a map of plasmid pAAVRSVF9;

FIG. 16 is a map of plasmid pCMVMBP-rep78;

FIG. 17 is a map of plasmid pAvALAPH81; and

FIG. 18 is a map of pAAVRSVApoF8.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an aspect of the present invention, there is provideda composition for delivering a DNA sequence encoding a protein orpolypeptide or genetic transcript of interest to a cell. The compositioncomprises an adeno-associated virus rep protein, or a nucleic acidsequence (DNA or RNA) encoding an adeno-associated virus rep protein.The composition also comprises a genetic construct which includes a DNAsequence encoding a protein or polypeptide or genetic transcript ofinterest; a promoter controlling the DNA sequence encoding a protein orpolypeptide or genetic transcript of interest; a first AAV ITR orportion or derivative thereof; and a second AAV ITR or portion orderivative thereof. The first and second adeno-associated viral ITR's(or portions or derivatives thereof) flank the DNA sequence encoding aprotein or polypeptide or genetic transcript of interest and thepromoter controlling the DNA sequence encoding the protein orpolypeptide or genetic transcript of interest.

In one embodiment, the adeno-associated virus rep protein is selectedfrom the group consisting of Rep 78, Rep 68, Rep 52, Rep 40, andfragments or derivatives thereof. The term “fragments or derivativesthereof” as used herein means that the rep protein may be a proteinwhich has deletion(s) of amino acid residues within the proteinstructure, and/or may be truncated at the C-terminal and/or theN-terminal, and/or may be mutated such that one or more amino acidresidues normally present in the protein structure are replaced withother amino acid residues. Such fragments and derivatives of repproteins that retain some or all of the same biological activities asthe unmodified rep proteins or that may possess modifiedcharacteristics.

In one embodiment, the adeno-associated virus rep protein is the Rep 78protein or a fragment of derivative thereof. In another embodiment, theadeno-associated virus rep protein is the Rep 68 protein or a fragmentor derivative thereof.

The adeno-associated virus rep protein may be produced by techniquesdisclosed in co-pending application Ser. No. 08/067,236, abandoned. Forexample, the rep protein may be synthesized on an automated proteinsynthesizer. Alternatively, the rep protein may be produced by geneticengineering techniques.

When the rep protein is produced by genetic engineering techniques, therep protein may be produced from cells transfected with an expressionvehicle including a nucleic acid sequence which encodes the rep protein.In one embodiment, the expression vehicle includes a first DNA sequenceencoding an adeno-associated virus rep protein or a fragment orderivative thereof, and a second DNA sequence encoding a protein or apeptide which is not an adeno-associated virus protein or peptide,whereby expression of said first DNA sequence and said second DNAsequence results in expression of a fusion protein including theadeno-associated virus rep protein or fragment or derivative thereof,and the protein or peptide which is not an adeno-associated virusprotein or peptide. The protein or peptide which is not anadeno-associated virus protein or peptide may be a bacterial protein orpeptide, or a histidine “tag” of 6 to 10 histidine residues.

In one embodiment, the protein or peptide which is not anadeno-associated virus protein or peptide is a bacterial protein. Thebacterial protein may be the E. coli maltose-binding protein, or afragment or derivative thereof. Maltose-binding protein, or MBP, has ahigh affinity for maltose and amylose. Fusion proteins which include MBPand rep protein can be isolated from lysates prepared from E. coli byadsorption and elution from an amylose affinity column. Thus, largequantities of AAV rep proteins can be isolated and purified, while suchAAV rep proteins retain their biological activities.

In another alternative, the rep protein is provided in an appropriateexpression vehicle containing a nucleic acid sequence (DNA or RNA)encoding the rep protein. The expression vehicle may be a plasmid vectorincluding the nucleic acid sequence encoding the rep protein.

The genetic construct, which includes a DNA sequence encoding a proteinor polypeptide or genetic transcript of interest, a promoter controllingthe DNA sequence encoding a protein or polypeptide or genetic transcriptof interest, and AAV ITRs or portions thereof which flank the DNAsequence and the promoter, is constructed such that the orientation ofthe AAV ITRs and the promoter is such that only the DNA sequenceencoding the protein or polypeptide or genetic construct of interest,and not any host genes, will be transcribed.

The AAV ITRs which flank the promoter and the DNA sequence controlled bythe promoter may be the complete ITR sequences or portions of the ITRsequences, which provide sufficient AAV ITR sequence to facilitatetargeted integration by rep protein. In one embodiment, the geneticconstruct includes at least the double-stranded oligonucleotidescontaining the AAV ITR A/A′ and D′/D regions, which are sufficient forthe rep functions believed to be needed for integration; non-covalentbinding, endonuclease action, covalent binding, helicase action, andrecruitment of host cell enzymes including DNA polymerases. An essentialfeature in the A/A′ region that facilitates non-covalent binding is theimperfect [GCTC]₄ repeat, and oligonucleotides can be constructed withalterations in this imperfect repeat sequence or in adjacent sequencesthat will effect non-covalent binding and/or nicking and covalentbinding. Alterations also may include modifications to the ITR hairpinsequences.

The genetic construct may be part of a plasmid, a fragment excised froma plasmid, a large synthetic oligonucleotide, or a “no-end” AAV DNA(i.e., a continuous strand of DNA joined at each end by the AAV ITRs,resulting in a continuous double-stranded DNA molecule).

In one embodiment, the DNA sequence which encodes a protein orpolypeptide of interest encodes a therapeutic agent. The term“therapeutic” is used in a generic sense and includes treating agents,prophylactic agents, and replacement agents.

DNA sequences encoding therapeutic agents which may be placed into thegenetic construct include, but are not limited to, DNA sequencesencoding tumor necrosis factor (TNF) genes, such as TNF-α, interferons,such as Interferon-α, Interferon-β, and Interferon-γ; genes encodinginterleukins such as IL-1, IL-1-B, and Interleukins 2 through 14; geneencoding GM-CSF; genes encoding adenosine deaminase or ADA; genesencoding cellular growth factors or cytokines, such as epithelial growthfactor (EGF), keratinocyte growth factor (KGF), and lymphokines, whichare growth factors for lymphocytes; gene encoding soluble CD4; FactorVII; Factor IX; T-cell receptors; the LDL receptor, ApoE, ApoC, ApoAI,and other genes involved in cholesterol transport and metabolism; thealpha-1 antitrypsin (α1AT) gene, the ornithine transcarbamylase gene,the CFTR gene, the insulin gene, negative selective markers or “suicide”genes, such as viral thymidine kinase genes, such as the Herpes SimplexVirus thymidine kinase gene, the cytomegalovirus thymidine kinase gene,and the varicella-zoster virus thymidine kinase gene; superoxidedismutase genes, such as Cu-SOD, Mn-SOD, and Zn-SOD; Fc receptors forantigen-binding domains of antibodies, and antisense sequences whichinhibit viral replication, such as antisense sequences which inhibitreplication of hepatitis B or hepatitis non-A non-B virus. Additionaltherapeutic agents include genetic transcripts such as a messenger RNA,antisense RNA, or ribozymes. It is to be understood, however, that thescope of the present invention is not intended to be limited to thespecific therapeutic agents described hereinabove.

The DNA sequence encoding the therapeutic agent may be the nativenucleic acid sequence which encodes the therapeutic agent or a fragmentor derivative of the native nucleic acid sequence which encodes afragment or derivative of the therapeutic agent, which retains the samebiological activity of the unmodified therapeutic agent, or an allelicvariant thereof. The term “allelic variant” as used herein means thatthe allelic variant is an alternative form of the native nucleic acidsequence which may have a substitution, deletion, or addition of one ormore nucleotides, which does not alter substantially the function of theencoded therapeutic agent. The DNA sequence may encode the full lengththerapeutic agent or may encode a fragment or derivative of thetherapeutic agent, and the DNA sequence may further include a leadersequence or portion thereof, a secretory signal or portion thereof ofthe gene encoding the therapeutic agent, and/or may further include atrailer sequence or portion thereof of the gene encoding the therapeuticagent.

The DNA sequence encoding the therapeutic agent is under the control ofa suitable promoter. Suitable promoters which may be employed include,but are not limited to, adenoviral promoters, such as the adenoviralmajor late promoter; or heterologous promoters, such as thecytomegalovirus (CMV) promoter; the Rous Sarcoma Virus (RSV) promoter;inducible promoters, such as the MMTV promoter, the metallothioneinpromoter; heat shock promoters; the albumin promoter; and the ApoAIpromoter. Alternatively, the gene may be under the control of its ownnative promoter. It is to be understood, however, that the scope of thepresent invention is not to be limited to any specific promoters.

The rep protein or DNA encoding the AAV rep protein and the geneticconstruct may be administered to a host in vivo or to eukaryotic cellsin vitro. In one embodiment, the rep protein is complexed with thegenetic construct and the complex of the rep protein and the geneticconstruct is introduced into eukaryotic cells in vitro viaelectroporation or an encapsulating medium such as a liposome or anadenovirus capsid. In another embodiment, the AAV rep protein (or anexpression vehicle including a nucleic acid sequence encoding AAV repprotein) and the genetic construct are encapsulated within a liposomeand administered in vivo to a patient, whereby the rep proteinfacilitates integration of the genetic construct into a human chromosomeat a defined chromosomal locus, Chromosome 19, 13.4-qter. The repprotein or expression vehicle including a nucleic acid sequence encodingrep protein, and the genetic construct may be encapsulated within theliposome or adenovirus capsid by means known to those skilled in theart. The use of a defined chromosome target minimizes the likelihood ofinadvertent inactivation of any host genes.

In one embodiment, the composition further includes a ligand which bindsto a desired cell type, tissue, or organ, or a ligand which isnon-tissue specific. Examples of ligands include, but are not limitedto, adenovirus pentons, fiber trimers, or inactivated adenovirions;fusogenic proteins derived from Sendai virus, Semliki forest virus, andinfluenza fusogenic peptides; asialoglycoprotein, which binds to theasialoglycoprotein receptor of liver cells; membrane bound cytokines;tumor necrosis factors (or TNF's) such as, for example, TNF-alpha andTNF-beta; transferrin, which binds to receptors on liver cells;Interleukin-2 which binds to receptors on activated T-cells, and neuraltissue cells; ApoB, which binds to the LDL receptor of liver cells;alpha-2-macroglobulin, which binds to the LRP receptor of liver cells;alpha-1 acid glycoprotein, which binds to the asialoglycoproteinreceptor of liver; mannose-containing peptides, which bind to themannose receptor of macrophages; sialyl-Lewis-X antigen-containingpeptides, which bind to the ELAM-1 receptor of activated endothelialcells; CD34 ligand, which binds to the CD34 receptor of hematopoieticprogenitor cells; CD40 ligand, which binds to the CD40 receptor ofB-lymphocytes; ICAM-1, which binds to the LFA-1 (CD11b/CD18) receptor oflymphocytes, or to the Mac-1 (CD11a/CD18) receptor of macrophages;M-CSF, which binds to the c-fms receptor of spleen and bone marrowmacrophages; circumsporozoite protein, which binds to hepatic Plasmodiumfalciparum receptor of liver cells; VLA-4, which binds to the VCAM-1receptor of activated endothelial cells; LFA-1, which binds to theICAM-1 receptor of activated endothelial cells; NGF, which binds to theNGF receptor of neural cells; HIV gp120 and Class II MHC antigen, whichbind to the CD4 receptor of T-helper cells; the LDL receptor bindingregion of the apolipoprotein E (ApoE) molecule; colony stimulatingfactor, or CSF, which binds to the CSF receptor; insulin-like growthfactors, such as IGF-I and IGF-II, which bind to the IGF-I and IGF-IIreceptors, respectively; Interleukins 1 through 14, which bind to theInterleukin 1 through 14 receptors, respectively; and the Fcantigen-binding domain of an immunoglobulin. The ligand may beconjugated to the rep protein or conjugated to a complex of rep proteinand the genetic construct or, when a liposome is employed to deliver therep protein and genetic construct, the ligand may be anchored in thephospholipid bilayer of the liposome.

The DNA sequence encoding the therapeutic agent is administered in anamount effective to produce a therapeutic effect in a host. The host maybe an animal host, and in particular a mammalian host. The mammalianhost may be a human or non-human primate. The exact dosage of DNA to beadministered is dependent upon various factors, including the age,weight, and sex of the patient, the type of genetic construct employed,the nature of the disorder to be treated, the type of AAV rep proteinemployed, the formulation of the lipid vesicle employed to deliver theDNA, and the type of cells to be transfected with the DNA.

In another embodiment, the composition of the present invention may beemployed in an animal model, wherein the composition of the presentinvention is administered to an animal in vivo. The animal is thenevaluated for expression of the therapeutic agent in vivo in order todetermine the effectiveness of a possible gene therapy treatment in ahuman patient.

Alternatively, the composition of the present invention, which includesa DNA sequence encoding a protein or polypeptide or genetic transcriptof interest, may be administered to eukaryotic cells, such as humancells, in vitro, whereby the cells are transfected with the geneticconstruct including the DNA sequence encoding the protein or polypeptideor genetic transcript of interest. In such an embodiment, the geneticconstruct may be administered in an amount of from about 0.75 μg toabout 1.5 μg of DNA per 5×10⁵ cells, preferably at about 1.25 μg of DNAper 5×10⁵ cells. The eukaryotic cells then may be administered to a hostas part of a gene therapy procedure, whereby such eukaryotic cellsexpress the protein or polypeptide or genetic transcript of interest ina host.

In another alternative, the composition of the present invention may beemployed to transfect eukaryotic cells in vitro, whereby suchtransfected eukaryotic cells are cultured in order to produce a desiredprotein or polypeptide of interest in vitro.

EXAMPLES

The invention now will be described with respect to the followingexamples; however, the scope of the present invention is not intended tobe limited thereby.

Example 1

A. Construction of pAAVRnLacZ

Plasmid AAVp5neo (Flotte, et al. , Am. J. Respir. Cell Mol. Biol., Vol.7, pgs. 349-356 (1992)) (FIG. 1) was cut with HindIII and KpnI to removethe neo^(R) gene, and the KpnI/BamHI fragment from pSV-βgalactosidase(Promega) (FIG. 2) was blunted and cloned into the blunted sites of theplasmid to form plasmid TRF169. (FIG. 3).

A second plasmid which provided the RSV-LTR promoter and nucleartargeting sequence for the lacZ gene was constructed as follows. TheBg1II/XbaI fragment containing the nlacZ gene from plasmid LZ11(Galileo, et al., Proc. Natl. Acad. Sci., Vol. 87, pgs. 458-462 (1990))(FIG. 4) was cloned into the blunted SmaI and BamHI sites of pSP72(Promega) (FIG. 5) to form pSP72nLacZ (FIG. 6). From pSP72nlacZ, theBg1II/BamHI fragment containing the nlacZ gene was removed and clonedinto the BamHI site of adRSV4 (FIG. 7) which was obtained from Dr.Beverly Davidson of the University of Michigan. The resulting plasmid isreferred to as pAdRSVnLacZ (FIG. 8).

pAAVrnLacZ (FIG. 9, ATCC No. 69492) was produced by inserting theSspI/DraIII fragment from pAdRSVnLacZ which contained the RSV-LTRpromoter, nuclear targeting signal linked to the lacZ gene into thePm1I/DraIII site of TRF169.

B. Preparation of AAV Rep Proteins

(i) Cloning of MBP-Rep 68Δ and MBP-Rep 78

The open reading frames of rep proteins Rep 68 and Rep 78 were generatedby PCR amplification. A common 5′ primer corresponding to nucleotides327-346 of adeno-associated virus (codons 3-9 of Rep 68 and the Rep 78open reading frame) was synthesized and used for both Rep 68 and Rep 78.Initially, Rep 68 was amplified using a 3′ primer corresponding to areverse complement of AAV nucleotides 2029-2048 (codons 570-576). PCRamplification was performed using cloned Pfu polymerase (Stratagene)with buffer. The PCR product was digested with HindIII, which cleavesAAV at nucleotide 1882, and ligated into plasmid pPR997 (FIG. 10) (NewEngland Biolabs), which was digested with XmnI and HindIII. Thus, a Rep68 gene was inserted into pPR997 in which 16 codons at the 3′ terminuswere deleted, thus resulting in the formation of a modified Rep 68protein, sometimes hereinafter referred to as Rep 68Δ, in which the last16 amino acids at the C-terminal have been deleted. pPR997 includes anE. coli malE gene, in which nucleotides 2-26 of the malE gene weredeleted, controlled by the E. coli tac promoter which includes anoperator site for the lacI repressor. pPR997 also includes a polylinkeror multiple cloning site. This cloning strategy resulted in the openreading frame of the Rep 68 gene ligating in frame with the malE openreading frame of pPR997 at the 5′ end of the Rep 68 gene. The 3′terminusof the Rep 68 gene is a frame-shifted fusion between the AAV Rep 68 openreading frame and the lacZα gene, resulting in an additional 50 residuesat the carboxy-terminus. The resulting plasmid is pMBP-Rep 68Δ. (FIG.11)

A mutant MBP-Rep 68Δ with a mutation in the putative nucleosidetriphosphate (NTP)-binding site was produced by substitution of aBamHI-HindIII fragment from the pHIV rep NTP plasmid with alysine-to-histidine mutation in codon 340 (K340H) (Owens, et al.,Virology, Vol. 184, pgs. 14-22 (1991); Owens, et al., J. Virol., Vol.67, pgs. 997-1005 (1993)) to form pMBP-Rep 68ΔNTP. (FIG. 12). MBP-Rep68Δ-NTP retains the DNA binding function of MBP-Rep 68Δ; however, otherbiochemical properties, such as helicase activity, are ablated.

pMBP-Rep 78 was generated by amplifying AAV nucleotides 1872-2239. Thissequence includes an overlapping region of Rep 68 and Rep 78 and the 3′terminus of Rep 78. The 5′ primer corresponds to AAV nucleotides1872-1894 and the 3′ primer corresponds to the reverse complement of AAVnucleotides 2215-2239, and also incorporates HindIII and XbaI sites. ThePCR product was digested with HindIII and ligated into HindIII digestedpMBP-Rep 68Δ. The resulting plasmid is pMBP-Rep 78. (FIG. 13)

The MBP-Rep 78 protein is an in-frame fusion protein between the malEopen reading frame and the adeno-associated virus open reading framebeginning at codon 3 of the Rep 78 gene. The 3′-terminus utilizes thenaturally occurring stop codon of the rep gene, and therefore there areno non-viral carboxy terminus residues.

(ii) Protein Expression

E. coli organisms were transfected with pMBP-Rep 68Δ NTP or pMBP-Rep 78according to standard techniques. The DNA encoding MBP-Rep 68Δ NTP orMBP-Rep 78 is under the control of the E. coli tac promoter which isrepressed by the lacI repressor gene product. Addition of IPTG preventsbinding of the lac repressor to the tac promoter, thereby enabling highlevels of expression of MBP-Rep 68Δ NTP or MBP Rep 78. Recombinants thatwere positive for the correct insert and orientation were screened forexpression of fusion protein. The bacterial clones that produced aprotein of the predicted molecular weight were grown on a larger scale.

One liter cultures of bacteria transformed with pMBP-Rep 68Δ NTP orpMBP-Rep 78 were obtained. A bacterial pellet was obtained from eachculture by centrifugation, and each bacterial pellet was resuspended in0.05 vol. of column buffer (200 mM NaCl, 20 mM Tris-Cl (pH 7.4), 1 mMEDTA, and 1 mM dithiothreitol). The bacteria were lysed by sonication byfour 30 second pulses. The suspension was cleared by centrifugation at9,000×g for 20 min. at 4° C.

The supernatant was loaded onto a column packed with amylose-Sepharoseresin equilibrated in column buffer. The column then was washed with 10column volumes of column buffer. The proteins then were eluted with1×column buffer containing 10 mM maltose. Approximately 1 ml fractionswere collected and 2 μl aliquots were analyzed by SDS-polyacrylamide gelelectrophoresis on an 8% SDS-polyacrylamide gel. The overall yield ofMBP-Rep 68Δ NTP or MBP-Rep 78 from a one-liter culture was from 4 to 12mg of protein.

C. Preparation of Liposomes Containing AAV Rep Protein and pAAVRnLacZ

Liposomes were made by mixing 3 μl of lipid in 25 μl of Optimem (Gibco)with 25 μl of an Optimem solution containing the plasmid pAAVRnLacZ at aconcentration of 0.05 μg/μl (yielding 1.25 μg DNA). The Optimem solutioncontaining the plasmid was preincubated for ½ hour at 37° C. with either(i) MBP-Rep 78 in amounts of 0.98 μg, 0.42 μg, 0.26 μg, or 0.19 μg; (ii)MBP-LacZ in amounts of 1.5 μg, 0.15 μg, or 0.015 μg; or (iii) MBP-Rep 68Delta NTP in amounts of 1.5 μg, 0.15 μg, or 0.015 μg.

D. Transfection of Cells with Liposomes Including AAV Rep Protein andpAAVRnLacZ

One-half hour after the solutions were mixed to form liposomes, thesolution containing the liposomes was added to human hepatoma derived,Hep G-2 cells that had been washed with PBS and then covered with aminimal volume of Optimem. The liposomes were added in an amount suchthat 1.25 μg of total plasmid DNA was added per 5×10⁵ cells. Prior tocontact of the cells with the liposomes, the cells had been grown inDMEM with 10% fetal calf serum and 2 mM glutamine. The cells were grownin an incubator having a 5% CO₂ atmosphere, and at 37° C.

Eighteen hours after the liposomes were added to the cells, serumcontaining medium was added. Thirty-three hours after liposome addition,the cells were washed with PBS, trypsinized and serum-containing mediumwas added to stop the trypsin action, and the cells were transported onice for cell cytometry.

To determine the optimal ratio between rep protein and a 5′ labeled AAVITR, experiments had been conducted using a covalent linkage assay. Theassay is dependent on three rep protein functions (non-covalent binding,endonuclease activity, and covalent binding). (Im, et al., J. Virol.,Vol. 63, pgs. 3095-3114 (1989); Chiorini, et al., J. Virol., Vol. 68,pgs. 7448-7457 (1994)). Maximal covalent bond formation occurred whenthe MBP Rep 78/ITR molar ratio was 9:1.

Using this information, cytometry was conducted at 36 hourspost-transfection. Cells were counted and viability was determined bytrypan blue and propidium iodide staining. Greater than 95% of the cellswere viable, which demonstrated that liposomes containing rep proteinwere not toxic. Cells expressing lacZ were detected by the use of thefluorescent B-galactosidase substrate fluorescein di-Betagalactopyranoside (FDG) and sorted into positive (+) and negative (−)populations. Non-viable cells were excluded from all % (+)determinations and sorting. Cytometry also was done to verify the purityof the sorted populations. This showed the sorted populations to begreater than 99% pure.

The percentage of positive cells (i.e., cells which expressed the lacZgene) for Hep G-2 cells treated with MBP-Rep 78 is given in Table Ibelow.

TABLE I MBP-Rep78/1.25 μg plasmid DNA/5 × 10⁵ cells picomoles moles Rep78/ μg Rep 78 Rep 78 moles plasmid % (+) 0.98 8.5 34 49 0.42 3.6 14 430.26 2.3 9 7 0.16 1.7 7 11 0 0 0 1

The percentage of positive cells (i.e., cells which expressed the lacZgene) at 36 hours post-transfection for HepG-2 cells treated withMBP-lacZ is given in Table II below.

TABLE II MBP-lacZ/1.25 μ g plasmid/5 × 10⁵ cells moles lacZ/ μg lacZpico moles lacZ moles plasmid % (+) 1.5 33 132:1  0 0.15 3.3 13:1 00.015 0.3  1:1 22

The percentage of positive cells which expressed the lacZ gene at 36hours post-transfection for HepG-2 cells treated with MBP-Rep 68-deltaNTP is given in Table III below.

TABLE III MBP-Rep 68 delta NTP/1.25 μg plasmid DNA/5 × 10⁵ cells molesRep 68- μg Rep 68- pico moles Rep delta NTP/moles delta NTP 68-delta NTPplasmid % (+) 1.5 14 56:1 0.5 0.15 1.4 5.6:1  35 0.015 0.14 0.56:1   35

Based on these results, MBP-Rep 78 appeared to cause earlier expressionof the lacZ gene in a dose-related fashion. This was a specific repprotein effect, because MBP-lacZ, a protein with no rep proteinfunctions, had the opposite effect, tending to show fewer positive cellswith increasing amounts of the protein. The MBP-Rep 78 effect appearedto require rep protein activities other than DNA binding, since MBP-Rep68 delta NTP, a mutant rep protein able to bind non-covalently but notnick AAV ITRs, had the same effect as MBP-lacZ.

The cells were maintained in culture for approximately 2 months and thensent for repeat cytometric analysis. Cell viability remained high. Thecells then were preincubated with chloroquine to reduce any backgroundpositivity, and then were stained with fluoroscein-di-Betagalactopyranoside (FDG) as hereinabove described. Non-viable cells wereexcluded by propidium iodide and cytometry, and the percent positivecells was determined for viable cells.

The percentage of positive cells (i.e., cells which expressed the lacZgene) at 2 months after transfection among cells that were positive at36 hours after transfection, and which were treated with MBP-Rep 78, isgiven in Table IV below.

TABLE IV MBP-Rep 78/1.25 μg DNA/5 × 10⁵ cells μ moles Rep 78/ μg Rep 78moles plasmid % (+) 0.98 34:1 10 0.42 14:1 8 0.26   9:1 18 0.19   7:1 130.00 0 7

The percentage of positive cells at 2 months after transfection amongcells that were negative at 36 hours after transfection, and which weretreated with MBP-Rep 78, is given in Table V below.

TABLE V MBP-Rep 78/1.25 μg DNA/5 × 10⁵ cells μ moles Rep 78/ μg Rep 78mles plasmid % (+) 0.98 34:1 80 0.42 14:1 53 0.26   9:1 22 0.19   7:1 220 0 5

The results of Tables IV and V indicated a dose-dependent MBP-Rep 78effect that was greatest with cells which originally had been negative.These cells had a high percentage of FDG-positive cells, and thispercentage increased with increasing dosage of Rep 78 employed. Thecells which had been positive at 36 hours post-transfection showed amuch lower percentage of stable positive cells and while Rep 78 appearedto increase long term percentage of positive cells, there was no doseeffect.

These results indicate that MBP-Rep 78, when delivered via a liposomewith an ITR-flanked gene, appears to augment, in a dose-dependentfashion, long-term transgene expression in cell culture. Suchaugmentation of expression is unlikely to have resulted from enhancedintegration, as it is unlikely for an extrachromosomal element topersist in culture for two months without continuous positive selectionpressure. From the above results, rep protein delivered by liposomeappears to have two effects in a cell: (i) enhancement of initialexpression of the transgene with a reduced effect on integration(initially positive cells that subsequently had lower levels ofpersistently positive cells); or (ii) suppression of initial transgeneexpression while facilitating integration (initially negative cells thatsubsequently had high levels of long-term positive cells.).

Example 2

Ex vivo treatment of Hemophilia B Using an AAV Vector for Human FactorIX

pAv1H9FR (FIG. 14, which includes an adenovirus 5′ ITR, an RSV promoter,a tripartite leader sequence, the 5′ untranslated region of the humanFactor IX gene, a centrally truncated first intron, the human Factor IXcoding region, the 3′ untranslated region of the human Factor IX gene, apolyadenylation signal, and an adenovirus homologous recombinationregion), is digested with SpeI and BamHI to obtain a fragment includingthe human Factor IX gene and the above-mentioned genomic elements andpolyadenylation signal. The fragment is blunt-ended with Klenow, andcloned into the ITR containing fragment of the NotI-BsmI digest ofpAAVrnlacZ to obtain pAAVRSVF9. (FIG. 15).

The plasmid pAAVRSVF9 contains the gene for Human Factor IX under thecontrol of the RSV promoter. There are 5′ and 3′ flanking AAV ITR's:

5′—AAV ITR—RSV Promoter—Human Factor IX gene—Poly A—AAV ITR—3′

A patient with Hemophilia B undergoes a partial hepatectomy usingappropriate coagulation factor support. The removed cells are placedinto culture at 37° C. One day later the cells are gently washed with IXPBS followed by gentle rewashing with Optimem (Gibco). The cells arecovered with a thin layer of Optimem. DNA containing liposomes areadded, such that there is a ratio of 1.25 micrograms of DNA added per5×10⁵ cells.

The liposomes are formed by mixing 3 microliters of lipid in 25microliters of Optimem (Gibco) with 25 microliters of an Optimemsolution containing the plasmid pAAVRSVF9 at a concentration of 0.05micrograms/microliter (yielding 1.25 μg DNA). The Optimem solutioncontaining the plasmid was preincubated for ½ hour at 37° C. with 1 μgof MBP-Rep 78.

Eighteen hours following the addition of the liposomes, serum containingmedium is added to the cells. On the following day the cells are putinto solution using standard trypsinization technique and reinfused intothe patient using an indwelling portal vein catheter placed at the timeof the initial partial hepatectomy. The catheter is removed followingthe reinfusion of the cells. The hepatic cells reingraft and producehuman Factor IX, thereby ameliorating the patient's Factor IXdeficiency.

Example 3 In Vivo Treatment of Hemophilia B Using Portal Vein Infusionof Liposomes

A lipid that is stable in the presence of serum is used to formliposomes. The liposomes are made by first mixing 5 microliters of thelipid in 25 microliters of a solution appropriate for formation of invivo liposomes. This lipid solution is then mixed with 25 microliters ofa solution containing 1.25 micrograms of the plasmid pAAVRSVF9 and 1microgram of either MBP-Rep 78 or a plasmid that contains the gene forRep 78. The solution used is one appropriate to the formation ofliposomes that can be used in vivo. pCMVMBPRep78 (FIG. 16) is an exampleof a plasmid that contains the gene for Rep 78. pCMVMBPRep78 wasconstructed as follows:

Using PCR, the ATG sequence located adjacent to the EcoRV site in the 5′untranslated region of pMBP rep 78 was changed to AGT. Theoligonucleotides used in this PCR had the sequences:

5′-ATATCAATTCACACAGGAAACG-3′ and

5′-GTTCGAATAGATCTTCTATTGG-3′.

The resultant plasmid, pMBPAGTRep78, then was digested with EcoRV andXbaI. The MBP-Rep 78 fragment then was cloned into the plasmid pCDNA,which had been opened with EcoRV and XbaI. The resultant plasmid,pCMVMBPRep78 (FIG. 16) has the gene for MBP-Rep 78 under the control ofthe CMV promoter.

If the MBP-Rep 78 protein is used to form the liposome, then the DNA andthe protein are preincubated at 37° C. in the Optimem solution for ½hour prior to mixing with the lipid-Optimem mixture. The liposomes arethen delivered to the liver of a patient with Hemophilia B using aportal vein catheter. The catheter is placed on the day of infusionusing appropriate coagulation factor support. A rough estimation of thenumber of hepatocytes that the patient has is made given his/her bodymass index, and liposomes diluted in sterile saline are infused suchthat approximately 1.25 micrograms of DNA are administered per 5×10⁵hepatocytes. The portal vein catheter is removed and the patient takenfor appropriate post-surgical care. Following uptake, those cells stablytransduced begin production of human Factor IX, thereby ameliorating thepatient's coagulation factor deficiency.

Example 4 In Vivo Treatment of Hemophilia B Using Liposomes andHepatic-selective Ligands

Liposomes are formed as described in Example 3, except that a hepaticselective ligand, such as asialoglycoprotein, is anchored in themembrane phase of the liposome. The ligand can be incorporated at thetime of liposome formation using an appropriate lipid tail connected tothe ligand, or the ligand can be incorporated following formation of theliposomes. In the latter case, the ligand can be attached to themembrane using any of a variety of standard techniques, includingcovalent chemical bond formation between the ligand and a membrane boundprotein. The liposomes are administered intravenously in a patient withHemophilia B. As the liposomes travel through the systemic circulationthey are selectively taken up by hepatocytes because the ligand binds toa receptor on the hepatocyte surface. This leads to hepatic-specificuptake and hence expression of the human Factor IX gene, therebyameliorating the patient's coagulation deficiency.

Example 5 Ex Vivo Treatment of Hemophilia A Using an AAV Vector forHuman Factor VIII and Human Endothelial Cells Reimplanted Using anOsmotic Pump

pAvALAPH81 (FIG. 17, which includes an adenoviral 5′ ITR, an albuminpromoter, and ApoA1 transcription initiation site, a human Factor VIIIcoding sequence, and an adenovirus homologous recombination fragment)was digested with SalI and ClaI to obtain an ApoAI—Factor VIII fragment.The fragment is blunt-ended with Klenow, and then blunt cloned intopAAVRSVF9. The pAAVRSVF9 plasmid is opened by digestion with EcoRV andClaI to remove the Factor IX—poly A portion of the plasmid. Theremaining fragment contains the AAV ITR's and the RSV promoter. Theresult of the pAvALPH81 cloning into pAAVRSVF9 is pAAVRSVApoF8 (FIG.18).

Endothelial cells are isolated from the veins of a patient withHemophilia A (Factor VIII deficiency) and maintained in culture at 37°C. Liposomes are formed as follows: 1.5 μg of plasmid pAAVRSVApoF8 and1.2 μg of MBP-Rep 78 are added to Optimem solution (Gibco) to yield atotal volume of 25 microliters. This mixture is gently triturated andincubated at 37° C. for ½ hour. Following incubation, the DNA-Repsolution is mixed with a solution that consists of 3 microliters oflipid in 25 microliters of Optimem. The resultant mixture is gentlytriturated and allowed to sit for ½ hour at room temperature.

The endothelial cells are gently washed with 1X PBS and then gentlyrewashed two times with Optimem. For every 10⁵ cells, 25 microliters ofliposomes are added. The cells are returned to a 37° C. incubator for 12hours, and then serum-containing medium is added. At 4 weeks followingthis procedure Factor VIII production by the cells is verified. Thecells are put into solution by standard trypsinization followed byinactivation of the trypsin with serum-containing medium. The cells areseeded onto the inner surface of the tubing of an osmotic pump deviceand the pump is implanted subcutaneously in the forearm of the patient.The cells in the pump produce Human Factor VIII and this proteindiffuses from the osmotic pump into surrounding tissues. It is thentaken up into the patient's bloodstream, correcting the Factor VIIIdeficiency.

The disclosure of all patents, publications, including published patentapplications, and database entries referenced in this specification arespecifically incorporated by reference in their entirety to the sameextent as if each such individual patent, publication, and databaseentry were specifically and individually indicated to be incorporated byreference.

It is to be understood, however, that the scope of the present inventionis not to be limited to the specific embodiments described above. Theinvention may be practiced other than as particularly described andstill be within the scope of the accompanying claims.

What is claimed is:
 1. A composition for delivering a DNA sequence to acell through integration of said DNA into a chromosome of said cell,wherein said composition does not generate viral particles, and whereinsuch composition comprises genetic material consisting essentially of:(a) a first genetic construct for integration into a chromosome of saidcell, said first genetic construct being free of DNA encodingadeno-associated virus rep protein and including, in a 5′ to 3′direction, a first adeno-associated viral ITR; a promoter controlling aDNA encoding a polypeptide or genetic transcript of interest and a DNAencoding a polypeptide or genetic transcript of interest; and a secondadeno-associated viral ITR; and (b) is a second genetic construct thatprovides, upon expression, an adeno-associated virus rep protein intrans with respect to said first genetic construct, wherein saidadeno-associated virus rep protein is the only adeno-associated viralprotein expressed by said second genetic construct.
 2. The compositionof claim 1, wherein said adeno-associated virus rep protein is selectedfrom the group consisting of Rep 78, Rep 68, Rep 52, Rep 40 andfragments or derivatives thereof that retain some or all of the samebiological activities as the unmodified Rep 78, Rep 68, Rep 52 or Rep40.
 3. The composition of claim 2, wherein said adeno-associated virusRep protein is the Rep 78 protein or fragment or derivative thereof thatretains some or all of the same biological activities of the unmodifiedRep 78 protein.
 4. The composition of claim 1, wherein said polypeptideof interest is a therapeutic agent.
 5. The composition of claim 1, andfurther comprising an encapsulating medium.
 6. The composition of claim5, wherein said encapsulating medium is a liposome.
 7. The compositionof claim 6, wherein said liposome further contains a ligand specific fora desired target cell, tissue, or organ.
 8. An isolated eukaryotic celltransfected with the composition of claim
 4. 9. A process fortransducing a cell in vitro with a DNA sequence, which process comprisescontacting said cell with genetic material coding for products that donot generate viral particles, wherein said genetic material consistsessentially of: (a) a first genetic construct for integration into achromosome of said cell, said first genetic construct being free of DNAencoding adeno-associated virus rep protein and including, in a 5′ to 3′direction, a first adeno-associated viral ITR, a promoter controlling aDNA encoding a polypeptide or genetic transcript of interest and a DNAencoding a polypeptide or genetic transcript of interest; and a secondadeno-associated viral ITR; and (b) a second genetic construct thatprovides, upon expression, an adeno-associated virus rep protein intrans with respect to said first genetic construct and wherein said cellis transduced with said first genetic construct and said second geneticconstruct upon contact of said cell with said first genetic constructand said second genetic construct, wherein said adeno-associated virusrep protein is the only adeno-associated viral protein expressed by saidsecond genetic construct.
 10. A composition for delivering a DNAsequence to a cell through integration of said DNA into a chromosome ofa cell, wherein said composition does not generate viral particles, andwherein said composition comprises: (a) genetic material, wherein saidgenetic material consists essentially of a genetic construct forintegration into a chromosome of said cell, said genetic construct beingfree of DNA encoding adeno-associated virus rep protein and including,in a 5′ to 3′ direction, a first adeno-associated viral ITR; a promotercontrolling a DNA encoding a polypeptide or genetic transcript ofinterest and a DNA encoding a polypeptide or genetic transcript ofinterest; and a second adeno-associated viral ITR; and (b) anadeno-associated virus rep protein, said adeno-associated virus repprotein being provided in trans with respect to said genetic construct,wherein said adeno-associated virus rep protein is the onlyadeno-associated viral protein included in said composition.
 11. Aprocess for transducing a cell in vitro with a DNA sequence, whichprocess comprises contacting said cell with (a) genetic material codingfor products that do not generate viral particles, wherein said geneticmaterial consists essentially of a genetic construct for integrationinto a chromosome of a cell, said genetic construct being free of DNAencoding adeno-associated virus rep protein and including, in a 5′ to 3′direction, a first adeno-associated viral ITR; a promoter controlling aDNA encoding a polypeptide or genetic transcript of interest and a DNAencoding a polypeptide or genetic transcript of interest; and a secondadeno-associated viral ITR; and (b) an adeno-associated virus repprotein provided in trans with respect to said genetic construct, andwherein said cell is transduced with said genetic construct upon contactof said cell with said genetic construct and said rep protein, whereinsaid rep protein is the only adeno-associated viral protein which ispresent as a result of said process and which contacts said cell. 12.The composition of claim 10, wherein said adeno-associated virus repprotein is selected from the group consisting of Rep 78, Rep 68, Rep 52,Rep 40 and fragments or derivatives thereof that retain some or all ofthe same biological activities as the unmodified Rep 78, Rep 68, Rep 52or Rep
 40. 13. The composition of claim 12, wherein saidadeno-associated virus Rep protein is the Rep 78 protein or fragment orderivative thereof that retains some or all of the same biologicalactivities of the unmodified Rep 78 protein.
 14. The composition ofclaim 10, wherein said polypeptide of interest is a therapeutic agent.15. An isolated eukaryotic cell comprising the composition of claim 14.16. The composition of claim 10, further comprising an encapsulatingmedium.
 17. The composition of claim 16, wherein said encapsulatingmedium is a liposome.
 18. The composition of claim 17, wherein saidliposome further contains a ligand specific for a desired target cell,tissue, or organ.